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Biomedical subjects

D Baltimore

Publications and source records attributed to D Baltimore.

At least 343 records · Page 19Linked to original sources
PubMed1981

Inhibition of transcription factor activity by poliovirus.

To study the poliovirus-induced inhibition of host-cell RNA synthesis, we prepared transcription extracts from mock-infected and poliovirus-infected HeLa cells. In contrast with the control extracts, poliovirus-infected cell extracts prepared 3 hr after infection were unable to transcribe specifically DNA templates recognized by RNA polymerase II. Accurate transcription by RNA polymerase III, however, was only slightly reduced. Supplementation of the infected cell extract with a crude preparation of transcription factors (S100) restored its ability to transcribe a polymerase II template specifically; supplementation with purified polymerase II had no effect. When the S100 was fractionated on a phosphocellulose column, the restoration activity eluted between 0.35 M and 1 M KCl. When we tested infected extracts for inhibitory activity by mixing uninfected and infected cell extracts, no in vitro inhibition of polymerase II transcription by the uninfected extract was evident. These results indicate that at least one factor required for specific transcription by polymerase II is deficient in extracts from poliovirus-infected cells.

HeLa Cells↗
PubMed1981

Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes.

The structure of immunoglobulin-related gene was analyzed in individual Abelson murine leukemia virus (A-MuLV)-transformed lymphoid cell lines. Essentially all of these lines, whether immunoglobulin-containing or null, had DNA rearrangements in the vicinity of the JH regions on both chromosomes as well as deletions of at least 5 kb of DNA 5' to JH. None of these lines, however, except rare light chain producers, had detectable rearrangement at either their kappa or lambda light chain loci. In contrast to A-MuLV-transformants derived from bone marrow. Those from early fetal liver frequently contained more than two and sometimes 12 or more distinct, rearranged JH-containing fragments. Cellular subclones derived from these lines had a subset, usually two, of the fragments found in the parent line. Therefore, heavy chain gene rearrangement appears to precede that of light chain gene rearrangement and is still continuing in certain cultured A-MuLV transformants.

Abelson murine leukemia virus↗
PubMed1981

Synthesis of immunoglobulin mu chain gene products precedes synthesis of light chains during B-lymphocyte development.

Immunoglobulin (Ig) gene expression has been followed during the later stages of development of the murine fetal liver. Biosynthetic labeling and immunoprecipitation were used to isolate Ig-related polypeptides from fetal and neonatal livers. By examination of the specific immune precipitates, the earliest detectable Ig was shown to consist only of mu heavy chain. At about the time of birth, when light chain synthesis became evident, separation of surface Ig-positive cells from surface Ig-negative cells by using anti-Ig-coated dishes showed that cells lacking surface Ig (pre-B lymphocytes) synthesized only mu chains. Thus, commencement of light chain synthesis was closely coordinated with the appearance of surface Ig. Ig RNA species were examined by electrophoretic fractionation and hybridization with cloned Ig DNA sequences. The sizes and amounts of Ig mRNA were found to correlate with the pattern of mu and light chain protein biosynthesis. mu chain RNA species appeared earlier in gestation than light chain RNA did, and only after birth did light chain sequences reach levels equivalent to those of mu chain. Cell populations enriched in pre-B lymphocytes also contained an excess of mu over light chain mRNA.

Animals↗
PubMed1981

Multiple differences between the nucleic acid sequences of the IgG2aa and IgG2ab alleles of the mouse.

To compare the structure of IgG2a alleles we have determined the complete DNA sequence of the constant region, coding sequence, and 3' untranslated region of a cDNA clone, pAB gamma 2a-1, which was derived from the C57BL/6 mouse strain (b allotype). This sequence was compared with the corresponding IgG2a DNA sequence of BALB/c origin (a allotype). The DNA sequences showed 10% differences, and the deduced protein sequences differed by about 15%. These differences were not evenly distributed: most differences were in the hinge region, the CH3 domain and the 3' untranslated region. It is evident that many alterations in the IgG2a alleles have occurred since the a and b haplotypes were separated--some of these changes were point mutations but some appear to have resulted from gene conversion of the IgG2ab allele by the IgG2bb allele.

Alleles↗
PubMed1981

Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome.

The complete 7410 nucleotide sequence of poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.

Cloning, Molecular↗
PubMed1981

Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase.

A rapid assay for retroviral reverse transcriptase activity released into the culture medium by infected cells was developed. With the assay, 4,000 clonally infected cell lines could be tested in a few hours. We have adapted the assay for use as a screen for the detection of spontaneous viral mutants. Mutants of Moloney murine leukemia virus have been isolated which (i) produce a thermolabile reverse transcriptase, (ii) are temperature sensitive for release of enzyme activity, or (iii) can only productively infect cells already producing gag-related polypeptides. The assay has also been useful for the isolation of nonproducer cells infected with various replication-defective transforming viruses.

Culture Media↗
PubMed1981

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

Abelson murine leukemia virus↗
PubMed1981

Genome structure of Abelson murine leukemia virus variants: proviruses in fibroblasts and lymphoid cells.

We have prepared full-length DNA clones of the Abelson murine leukemia virus (A-MuLV) genome. A specific probe homologous to the central portion of the A-MuLV genome was prepared by nick translation of a subcloned restriction fraction from the cloned DNA. The probe was used to examine the genome structure of several A-MuLV variants. The conclusions are: (i) three viruses coding for Abelson-specific proteins of molecular weight 120,000, 100,000, and 90,000 had genomes indistinguishable in size, suggesting that the shorter proteins are the result of early translational termination; (ii) compared with the genome encoding the 120,000-dalton (120K) protein, a genome coding for a 160K protein was 0.8 kilobase larger in the A-MuLV-specific region; and (iii) a genome coding for a 92K protein had a 700-base pair deletion internal to the coding region. This mutant was transformation defective: its 92K protein lacked the protein kinase activity normally associated with the A-MuLV protein, and cells containing the virus were not morphologically transformed. In addition, we determined the number of A-MuLV proviruses in each of several transformed fibroblast and lymphoid cells prepared by infection in vitro. These experiments show that a single copy of the A-MuLV provirus is sufficient to transform both types of cells and that nonproducer cells generally have only one integrated provirus.

Abelson murine leukemia virus↗
PubMed1981

Phosphorylation of the Abelson murine leukemia virus transforming protein.

The Abelson murine leukemia virus transforming gene product is a phosphorylated protein encoded by both viral and cellular sequences. This gene product has an amino-terminal region derived from the gag gene of its parent virus and a carboxyl-terminal region of (abl) derived from a normal murine cellular gene. Using a combination of partial proteolytic cleavage techniques and antisera specific for gag and abl sequences, we mapped in vivo phosphorylation sites to different regions of the protein. Phosphoproteins encoded by strain variants and transformation-defective mutants of Abelson murine leukemia virus with defined deletions in the primary sequence of the abl region were compared by two dimensional limit digest peptide mapping. Specific phosphorylation pattern differences for wild-type and mutant proteins probably represented deletions of specific phosphate acceptor sites in the abl region. An in vitro autophosphorylation activity copurified with the Abelson murine leukemia virus protein from transformation-competent strains. A peptide analysis of such in vitro reactions demonstrated that these phosphorylation sites were restricted to the amino-terminal region, and the specific sites appeared to be unrelated to the sites found on proteins phosphorylated in vivo. Thus, the autophosphorylation reaction probably correlates with an activity important in transformation, but the specific end product in vitro bears little resemblance to its function in vivo.

Abelson murine leukemia virus↗
PubMed1981

Intramolecular integration within Moloney murine leukemia virus DNA.

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features.

Bacteriophage lambda↗
PubMed1981

Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix: architecture of the protein.

We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule.

Abelson murine leukemia virus↗
PubMed1980

Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA.

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.

Cell Nucleus↗
PubMed1980

Cytogenetic studies on abelson-virus-induced mouse leukemias.

The karyotype of Abelson-virus-induced murine leukemias was studied by G-banding. In contrast to the regular trisomy of chromosome 15 in most murine T-cell leukemias, Abelson leukemias were purely diploid, and remained diploid for up to seven consecutive passages in vivo. The hypothesis is advanced that integration into the recipient cell of the DNA copy of the large cellular insert, carried by the Abelson virus, may perform a function similar to the effects of gene duplication by trisomy in the more slowly developing murine leukemias.

Abelson murine leukemia virus↗
PubMed1980

Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine.

The Abelson murine leukaemia virus protein (P120) can become phosphorylated in vitro by [gamma-32P]ATP. The protein has been purified from cell membranes to the point that in specific conditions virtually all of the incorporated 32P is in P120. The reaction is stimulated by Mn2+ and Mg2+ but not Ca2+ and is very rapid even at 0 degrees C. The phosphate is linked to P120 at tyrosine, a linkage not previously reported for a phosphorylation reaction. Phosphorylation may be involved in the transforming activity of viruses that cause leukaemia as well as sarcomas.

Abelson murine leukemia virus↗
PubMed1980

Biosynthesis of murine terminal deoxynucleotidyltransferase.

An immunoprecipitation assay for measuring synthesis of murine terminal deoxynucleotidyltransferase (EC 2.7.7.31) has been developed using rabbit antiserum to calf terminal transferase. The antiserum precipitates a single Mr = 60,000 polypeptide (TdT-60) from all cell lines and tissues that contain enzymologically demonstrable terminal transferase. This polypeptide is not precipitated from labeled extracts of cells that lack terminal transferase by enzymological criteria. TdT-60 fractionates with terminal transferase during phosphocellulose chromatography and sediments with it in a sucrose gradient. TdT-60 is not detectably processed to lower molecular weight polypeptides, and terminal transferase activity sediments as a Mr = 60,000 activity; thus, we believe it to be the active form of terminal transferase. Using this assay we have demonstrated that terminal transferase is synthesized in both the murine thymus and the bone marrow at a rate proportional to its biochemically measured steady state level. After cortisone treatment of mice, the Mr = 60,000 polypeptide disappears from the thymus and then reappears as the thymus begins to be repopulated.

Animals↗